Methods of producing HBsAg

ABSTRACT

Hepatitis B surface antigen (HBsAg) is produced in vitro in high titer and purity from tissue cultures of cells that shed HBsAg. The cells are grown on hollow fiber capillary units having a molecular weight cut-off of about 10,000.

BACKGROUND OF THE INVENTION

Hepatitis B surface antigen (HBsAG) has been shown to be effective as avaccine against hepatitis B disease. The usual source of this antigen isplasma obtained from donors, e.g., by phasmaphoresis. As a result thesupply of plasma containing this antigen is uncertain and expensive asmost plasma is free of HBsAG.

It has been known heretofore to grow in vitro on hollow fiber capillaryunits tissue cultures of cells which shed HBsAG. The hollow fiber unitsused heretofore have had a molecular weight cut off of 100,000 orgreater.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide an improved in vitrotissue culture method for preparing HBsAG. Another object is to providea method for preparing HBsAG in higher yield and at a faster rate. Theseand other objects of the present invention will be apparent from thefollowing description.

SUMMARY OF THE INVENTION

Hepatitis B surface antigen (HBsAG) is produced in vitro in high titerand purity from tissue cultures of cells that shed HBsAG. The cells aregrown on hollow fiber capillary units having a molecular weight cut-offof about 10,000.

DETAILED DESCRIPTION

The present invention relates to a method of producing hepatitis Bsurface antigen (HBsAG) in vitro on hollow fiber capillary units havinga molecular weight cut-off of about 10,000.

It has now been found that the yield of HBsAG from cells which shedHBsAG is increased markedly and at a faster rate when the cells aregrown in cell culture in vitro on hollow fiber capillary units having amolecular weight cut-off of about 10,000. Higher yields are obtainedwhen the growth cycle has two different elevated temperatures. Eachtemperature is above room temperature and the first temperature ishigher than the second temperature. The first temperature is from about35° to about 38° C. while the second temperature is from about 30° toabout 34° C. Preferably the first temperature is about 37° C. and thesecond temperature is about 32° C.

It has been found in addition that higher yields are obtained whencaffeine is present in the in vitro cell culture nutrient medium in anamount effective to improve yield of HBsAG or when the in vitro cellculture is effected on a permeable membrane such as, for example, abundle of hollow fiber capillary units. The caffeine may be present in alevel at which it exerts a detectable improvement in yield up to a levelabove which it exerts a toxic effect. Typically the caffeine is employedat from about 0.0001 M to about 0.007 M, preferably at from about 0.0001M to about 0.0003 M.

Any cells which shed HBsAG may be used in the process of the presentinvention. Examples of such cells are some human hepatomas, highyielding clones from such human hepatomas, and liver cells fromhepatitis B infected chimpanzees.

The human hepatoma tissue is grown in vitro in the presence of anutrient medium. By a nutrient medium is meant one which permits growthof cells in vitro. Some specific nutrient media are, for example, Medium199, Morgan et al., Proc. Soc. Exp. Biol. & Med.; 73:1-8, 1950; BasalMedium Eagle, Eagle Science, 122, 501-504, 1955; Morton, In Vitro, Vol.6, No. 2, pp. 89-108, 1970; Dulbecco's Modified Eagle's Medium, Dulbeccoet al., Virology, 8, 396, 1959; Smith et al., J. Virol. 12, 185-196,1960; Morton, op. cit. Minimum Essential Medium (Eagle), Science, 130,432 (1959) and RPMI Media, Moore et al., J.A.M.A. 199, 519-524, 1967;Morton, op. cit.

The hollow fiber capillary unit is formed of a plurality of anisotropichollow fiber membranes which provide a matrix for the culture of cellsand organ explants. A bundle of these capillaries forms a threedimensional vascular system which permits controlled perfusion of cellaggregates with nutrients, as well as exchange of excreted substances.The capillaries consist of a sponge-like body with a very thin (0.5-1μm), smooth layer of extremely fine, controlled pore size (approximaterange: 0.001-0.01 μm) on the lumen side. From that surface outward, thepores become increasingly larger 5-10 μm at the perimeter. Thisstructure provides a unique combination of selectively and extremelyhigh permeability to liquids even at very low or zero pressure. Thechoice of desired membrane "cut-off" levels offers selective control ofmacromolecule transport. The open exterior of the capillaries presents alarge surface for cell attachment and allows cells to penetrate towardthe barrier of the lumen. The anisotropic fiber membranes may beprepared as described in U.S. Pat. No. 3,615,024. The bundle of hollowfiber capillary units may be prepared as described in U.S. Pat. No.3,821,087.

While many types of cells have been grown on hollow fiber capillaryunits, e.g., mouse fibroblasts, human breast and choriocarcinoma, ratpancreas, rat pituitary tumor, rat villus crypt, human hepatocytes, rathepatoma, moneky kidney, baby hamster ovary, and rat lung, due to themany variables in biologic materials, preparation and operatingparameters, specific performance with other types of cells cannot beforecast.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1 (comparative)

A unit of capillary bundles (Vitafiber® Amicon hollow-capillary unit3P10) having a 10,000 molecular weight cut-off point, a 1,000 mlreservoir bottle, a peristaltic pump and Silastic tubing (about 2meters) are autoclaved and assembled under aseptic conditions in thefollowing manner: ##STR1## The extracapillary space (2 ml) is chargedwith a suspension of 7.5×10⁶ cells of a freshly harvested human hepatomacell line (PLC/PRF/5, MacNab et al., Br. J. Cancer (1976) 34, 509-515, aculture of which has been deposited with American Type CultureCollection and given accession number CCL 8024. The unit is placed in a37° C. CO₂ incubator. Every 30 minutes the hollow fiber unit is turned180° around its longest axis. After 2 hours Eagle's Minimum EssentialMedium (EMEM) containing 10% fetal calf serum, L-glutamine 2 mM, andNeomycin 50 μg/ml is circulated through the capillary unit at a flowrate of 5 ml/minute. After 2 weeks at 37° C. the temperature of theincubator is reduced and maintained at 32° C. and 10⁻⁴ M caffeine isadded. Cell growth is monitored by glucose utilization. Antigen samplesare taken from the extracapillary space and assayed by complementfixation. The results are summarized in the following table:

    ______________________________________                                        Age of Cell Culture (days)                                                                     Complement Fixation Titer                                    ______________________________________                                        7                4                                                            14               16                                                           21               64                                                           28               512                                                          35               512                                                          ______________________________________                                    

Elimination of the fetal calf serum does not have any significant effecton titers and facilitates further purification of the HBsAG.Conventional monolayer tissue culture systems under similar conditionsproduced only traces of antigen.

EXAMPLE 2

Three units of capillary bundles (Vitafiber®, Amicon) one 3P10, one P30and one 3S100 having respectively molecular weight cut-off points of10,000; 30,000 and 100,000 are each charged with a suspension of 6.0×10⁶cells of a freshly harvested higher HBsAG yielding clone of PLC/PRF/5cells. The units, set-up as described in Example 1, are placed in a 37°C. incubator. After 2 hours Eagle's Minimum Essential Medium (EMEM)containing 10% fetal calf serum, L-glutamine 2 mM, and Neoymcin 50 μg/mlis circulated through the capillary unit at a flow rate of 5 ml/minute.After 2 weeks at 37° C. the temperature of the incubator is reduced andmaintained at 32° C. and 10⁴ M caffeine is added. Cell growth ismonitored by glucose utilization. Antigen samples are taken from theextracapillary space and assayed by complement fixation. Samples fromthe circulating fluid are assayed by an enzyme immunoassay (AUS2 YME™,Abbott Labs). The results are summarized in the following table:

    ______________________________________                                        Day    10,000 MW    30,000 MW 100,000 MW                                      ______________________________________                                        14     16           4         4                                               21     64           64        8                                               28     128          64        32                                              33     256          64        16                                              39     256          128       32                                              ______________________________________                                    

The unit with 10,000 molecular weight cut-off point proves superioryields although glucose consumption is similar in all three units.

What is claimed is:
 1. A method for preparing hepatitis B surfaceantigen which comprises growing cells which shed hepatitis B surfaceantigen in the presence of a nutrient medium on hollow fiber capillaryunits having a molecular weight cut-off of about 10,000.
 2. A methodaccording to claim 1 wherein the growing is effected with two sequentialstages having differing temperatures, each temperature being above roomtemperature with the first temperature being above the secondtemperature.
 3. A method according to claim 2 wherein the firsttemperature is from about 35° to about 38° C.
 4. A method according toclaim 2 wherein the second temperature is from about 30° C. to about 34°C.
 5. A method according to claim 2 wherein the first temperature isabout 37° C. and the second temperature is about 32° C.
 6. A methodaccording to claim 2 wherein caffeine is present during the secondstage.
 7. A method according to claim 6 wherein the caffeine is presentin an amount from about 0.0001 M to about 0.0003 M.